A Glu urea Lys Ligand conjugated Lipid Nanoparticle/siRNA System Inhibits Androgen Receptor Expression In Vivo Citation Molecular Therapy—Nucleic Acids (2016) 5, e348; doi 10 1038/mtna 2016 43 Officia[.]
Trang 1We have previously shown that inhibition of androgen recep-tor (AR) expression and reduction of prostate-specific antigen (PSA) serum levels in mouse models of human prostate can-cer (PCa) can be achieved by intravenous (i.v.) administra-tion of lipid nanoparticles (LNPs) containing small interfering RNA (siRNA) targeting the gene encoding the AR (LNP-AR-siRNA).1 However, relatively high doses (six doses at 10 mg siRNA/kg body weight) were required to see appreciable effects This is in significant contrast to the potency of LNP-siRNA systems that have achieved 50% silencing of a hepatic gene with a single dose of 0.005 mg siRNA/kg.2–4 This large-dose disparity is attributed to the liver’s favorable physiology and to endogenous processes that result in targeting of LNPs
to hepatocytes.3,5 In particular, LNPs associate with apolipo-protein E (ApoE)3,6 following i.v administration and are taken into hepatocytes through the LDL receptor, the scavenger receptor, and the “LDL-like” receptor.3,7 Although it is unlikely that potencies equivalent to those seen for gene silencing in hepatocytes can be achieved in localized and disseminated PCa, reductions in current dose levels must be achieved for this approach to become a viable clinical strategy
The objective of this study was to improve the potency of the LNP-siRNA system developed previously.1 The primary techniques that we explored concerned using a different polyethylene glycol (PEG)-lipid to achieve longer circulation
lifetimes and facilitate higher levels of LNP accumulation at tumor sites, as well as the use of targeting ligands attached
to the LNP-siRNA system to specifically enhance uptake into PCa cells following arrival at the tumor site However, in order
to build the most potent LNP system possible, two other LNP variables were investigated: first, the use of a more potent cationic lipid and second, a more potent AR-siRNA than used previously.1
The cationic lipid, DLin-KC2-DMA, used in our previous study1 was identified by screening a variety of cationic lipids
in LNP-siRNA systems.2 Recent advances in cationic lipid design have resulted in a number of more potent cationic lipids, including DMAP-BLP,8 which results in improved gene-silencing potency when used in LNP-siRNA formulations compared to DLin-KC2-DMA.4 Furthermore, the AR-siRNA used previously1 was a 25-mer siRNA complementary to nucleotides 3542-3563 in the AR mRNA region that encodes the ligand-binding domain Improvement in gene silencing can be expected with the use of other sequence-optimized siRNAs as it has been shown that siRNAs against differ-ent regions of an mRNA have drastically differdiffer-ent silencing activities.9,10
Additional factors that intrinsically influence the silenc-ing activity of LNP-siRNAs are their ability to accumulate at the target site and to be taken up into the target cells LNPs with long half-lives in circulation are likely to accumulate at tumors due to the impaired lymphatic drainage and leaky
Received 10 May 2016; accepted 11 May 2016; published online 16 August 2016 doi:10.1038/mtna.2016.43 2162-2531
e348
Molecular Therapy—Nucleic Acids
10.1038/mtna.2016.43
5
10May2016
11May2016
2016
Official journal of the American Society of Gene & Cell Therapy
PSMA-targeted Lipid Nanoparticle siRNA Systems
Lee et al.
The androgen receptor plays a critical role in the progression of prostate cancer Here, we describe targeting the prostate-specific membrane antigen using a lipid nanoparticle formulation containing small interfering RNA designed to silence expression of the messenger RNA encoding the androgen receptor Specifically, a Glu-urea-Lys PSMA-targeting ligand was incorporated into the lipid nanoparticle system formulated with a long alkyl chain polyethylene glycol-lipid to enhance accumulation at tumor sites and facilitate intracellular uptake into tumor cells following systemic administration Through these features, and by using
a structurally refined cationic lipid and an optimized small interfering RNA payload, a lipid nanoparticle system with improved potency and significant therapeutic potential against prostate cancer and potentially other solid tumors was developed Decreases in serum prostate-specific antigen, tumor cellular proliferation, and androgen receptor levels were observed in a mouse xenograft model following intravenous injection These results support the potential clinical utility of a prostate-specific membrane antigen–targeted lipid nanoparticle system to silence the androgen receptor in advanced prostate cancer.
Subject Category: Nanoparticles
The first two authors and the last two authors contributed equally to this work
1Department of Biochemistry and Molecular Biology at the University of British Columbia, Vancouver, British Columbia, Canada; 2Vancouver Prostate Centre, Vancouver, British Columbia, Canada; 3Department of Drug Discovery, Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA Correspondence: Pieter R Cullis, Deaprtment of Biochemistry and Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3 E-mail: pieterc@mail.ubc.ca
Keywords: androgen receptor; lipid nanoparticles; liposomes; prostate cancer; prostate specific membrane antigen; siRNA
A Glu-urea-Lys Ligand-conjugated Lipid Nanoparticle/
siRNA System Inhibits Androgen Receptor Expression
In Vivo
Justin B Lee 1 , Kaixin Zhang 2 , Yuen Yi C Tam 1 , Joslyn Quick 1 , Ying K Tam 1 , Paulo JC Lin 1 , Sam Chen 1 , Yan Liu 1 , Jayaprakash K Nair 3 , Ivan Zlatev 3 , Kallanthottathil G Rajeev 3 , Muthiah Manoharan 3 , Paul S Rennie 2 and Pieter R Cullis 1
Trang 2vasculature at these sites.11,12 The PEG-lipid used in the
pre-vious studies was anchored into the LNP formulation by two
C14 alkyl chains; these lipids rapidly exchange out of the LNP
with half-times on the order of minutes following i.v
injec-tion.13,14 This loss of the PEG-lipid results in short (< 1 hour)
LNP circulation half-times with substantial liver accumulation
In order to achieve longer circulation lifetimes, we examined
here the properties of LNP systems containing PEG-lipid with
long alkyl chains, which can reside in the LNP formulation for
24 hours or longer
The type of targeting ligand to actively target LNP-siRNA
systems to PCa cells is of interest A small
molecule-tar-geting approach has been previously demonstrated for
anisamide,15–17 as well as the cardiac glycoside strophanthidin
(STR).18 The prostate-specific membrane antigen (PSMA),
a plasma membrane glycoprotein that is overexpressed in
PCa cells as well as the neovasculature of many solid tumors
(but not in healthy tissues),19 represents an attractive target
for LNP systems Binding to PSMA results in internalization
through clathrin-mediated endocytosis and thus can
poten-tially carry LNP into the cell.20 In silico screening studies have
identified the small molecule
2-(3-(1,3-dicarboxypropyl)-ure-ido)pentanedioic acid (DUPA),21 which binds specifically to
PSMA with high affinity.21–23 Urea-based analogs of DUPA
have served as the template for further development of
vari-ous potent PSMA-targeting ligands and have been studied by
the Kozikowski,24,25 Spiegel,26–28 and low groups,21–23
result-ing in diagnostics and therapeutics that have considerable
clinical potential.29 In this manuscript, we achieved improved
potency in AR silencing by incorporating a DUPA analog for
PSMA-targeting in long-circulating LNP systems that contain
optimized cationic lipid and siRNA against AR
Results
Optimized LNP-AR21-siRNA silences AR expression
in vitro
Our first optimization of the LNP-AR-siRNA employed the
ionizable cationic lipid, DMAP-BLP,8 which is three times
more potent in hepatic gene silencing than the
DLin-KC2-DMA,4 the lipid used in earlier LNP-AR-siRNA studies.1
Furthermore, the previously used AR siRNA (AR25-siRNA)
was a 25-mer derived from an shRNA sequence that was
shown to mediate silencing of AR and tumor growth delay in
vivo.30 In order to improve gene-silencing activity, a 21-mer
siRNA (AR21-siRNA) was identified in a screen of siRNAs
targeting the AR gene (data not shown) We incorporated two
phosphorothioate linkages in order to reduce degradation by
serum nucleases such as ribonuclease A (RNase A) and
RNase A-like enzymes,31 as well as multiple 2′-OMe
modi-fications to enhance stability in the presence of nucleases
and to prevent undesired immune responses.32 To compare
relative potencies of the 25-mer and 21-mer siRNAs, LNPs
containing either AR21-siRNA or AR25-siRNA were
incu-bated with LNCaP cells in vitro at siRNA concentrations of
0.5, 1.0, or 5.0 μg/ml for 48 hours and AR protein levels were
analyzed by immunoblotting (Figure 1) Essentially, complete
AR silencing was observed in cells treated with LNP
con-taining AR21-siRNA at all dose levels tested, whereas AR
protein knockdown was incomplete in cells treated with all
doses of AR25-siRNA Untreated cells or cells treated with control siRNA (a scramble sequence or siRNA against glyc-eraldehyde 3-phosphate dehydrogenase (GAPDH) showed
no reduction of AR levels (Supplementary Figures S3 and S4) Alternatively, another siRNA sequence against the AR
also showed appreciable AR knockdown (Supplementary Figure S4) These results indicate that the AR21-siRNA is
a more potent sequence than AR25-siRNA for silencing the
AR gene in LNCaP cells in vitro In addition, an alternate
siRNA against The AR21-siRNA was used in all subsequent experiments
LNPs containing PEG-DSG accumulate in distal tumors
Previously reported studies of AR siRNA utilized LNPs containing PEG-DMG, a PEG-lipid with two C14 alkyl chains.1 PEG-lipids are required to produce LNP systems with defined sizes33 and to prevent aggregation of the LNP after formation.34 As noted elsewhere,13,14 PEG-lipids with
C14 chains rapidly dissociate from the LNP (dissociation
halftimes of minutes or less) following in vivo
administra-tion and result in short circulaadministra-tion lifetimes, enhancing liver accumulation but reducing LNP accumulation at tumor sites In order to improve tumor accumulation, LNP sys-tems used in this work incorporated PEG-DSG, a lipid with
C18 alkyl chains Previous work has shown that PEG-lipids with C18 alkyl chains remain associated with LNP for days
or longer,14 leading to extended circulation lifetimes follow-ing i.v administration relative to those with shorter PEG chains.13,14,35 Consistent with these reports,35 we found that LNP-siRNA, produced by microfluidic mixing incorporating 1.5% PEG-DSG, exhibited extended circulation properties compared to LNP with equivalent amounts of PEG-DMG.14
Increasing the total PEG-DSG lipid in the LNP from 2.5 to 5% resulted in a marked increase in circulation halftime from approximately 30 minutes to greater than 8 hours ( Fig-ure 5) To show that increased circulation lifetime translates
to enhanced LNP accumulation in tumors, fluorescently labeled LNP-AR21-siRNA containing either 2.5 or 5 mol% PEG-DSG were prepared and administered intravenously once every day for 3 days at a dose of 10 mg siRNA/kg body weight in athymic nude mice bearing LNCaP tumors Tumors were harvested at 4 and 24 hours following the final
Figure 1 Lipid nanoparticle (LNP) encapsulating AR21-siRNA results in enhanced AR knockdown in vitro AR21-siRNA was
composed of two complementary RNA strands: sense strand S) 5′-cuGGGAAAGucAAGcccAudTsdT-3′ and antisense strand (AR-AS): 5′-AUGGGCUUGACUUUCCcAGdTsdT-3′ LNCaP cells were incubated with 0.5, 1.0 or 5.0 μg/ml of LNP encapsulated with either AR25-siRNA or AR21-siRNA for 48 hours Equal portions of protein samples were analyzed by immunoblotting to AR and β-actin, the latter serving as a loading control
AR25-siRNA µg/ml siRNA Unt
AR
β-actin
AR21-siRNA
Trang 3injection of LNP formulations, fixed in 10% formalin,
cryo-sectioned, and analyzed for LNP accumulation by
confo-cal microscopy Consistent with the enhanced circulation
lifetime, tumor tissues from mice treated with LNP
contain-ing 5 mol% PEG-DSG showed significantly higher
accumu-lation of fluorescence than those from mice injected with
LNP containing 2.5 mol% PEG-DSG (Figure 2b)
Fluores-cent micrographs showed that LNPs accumulated in tumor
tissues over time, with higher levels observed in tumors
collected 24 hours postadministration than at 4 hours
Approximately fourfold more LNP containing 5 mol%
PEG-DSG was observed in tumor tissues than LNP containing
2.5 mol% PEG-DSG (Figure 2b)
Incorporation of (Glu-urea-Lys)-PEG-DSG into LNP systems results in increased cellular uptake and AR gene silencing in LNCaP cells and is enhanced via a PSMA-dependent endocytic mechanism
A durable PEG coating enhances LNP accumulation at sites
of tumors, but also hinders uptake into cells and subsequent delivery of the siRNA payload to the cytoplasm A strategy
to overcome this problem is to incorporate into the LNP a small-molecule homing ligand that targets a cell surface receptor.15,18 As indicated under Methods, we synthesized a small-molecule PSMA-targeting ligand and chemically conju-gated it to PEG-DSG, as shown in Figure 3
Our PSMA-targeting analog is based on a Glu-urea-Lys scaffold and was designed according to previously published data.21–23 The PSMA-targeting lipid (13, Figure 3) is com-posed of a Glu-urea-Lys moiety tethered to the PEG lipid through a phenyl ring We first synthesized the appropriately protected Glu-urea-Lys carboxylic acid 5 ( Figure 3) from
2-[3-(5-amino-1-tert-butoxycarbonylpentyl)-ureido]pentane-dioic acid di-tert-butyl ester (4)25 and from the carboxylic acid
3 under peptide coupling conditions, followed by selective
deprotection of the benzyl ester as shown in Scheme 1 The carboxylic acid 3 was synthesized from benzyl 5-hexynoate
(1)36 and (p-iodophenyl)acetic acid (2) by standard
Sono-gashira coupling.37 Covalent attachment of the protected car-boxylic acid (5) under peptide coupling conditions with the
amino-PEG-lipid (11) followed by deprotection of functional
groups afforded the desired (Glu-urea-Lys)-PEG-DSG lipid (13), used for PSMA-targeting LNP formulation of the siRNA
The experimental details and compound characterization are included in the Supplementary Materials and Methods.
To evaluate the effects of incorporating (Glu-urea-Lys)-PEG-DSG on cell uptake, fluorescently-labeled LNP systems were utilized A PSMA-targeted LNP-AR-siRNA system con-taining 1 mol % (Glu-urea-Lys)-DSG and 1.5% PEG-DSG was compared to an untargeted LNP system containing
a total of 2.5% PEG-DSG lipid Uptake into LNCaP cells was analyzed by fluorescence microscopy At 24 hours, fluo-rescence was approximately fourfold higher in cells treated with the PSMA-targeted LNP-AR-siRNA compared to those treated with the nontargeted LNP (Figure 4a)
To verify that uptake was via a PSMA-dependent mecha-nism, LNCaP cells were treated with (Glu-urea-Lys)-LNP
in the presence or absence of the competitive reagent, 2-PMPA, at 100-fold molar excess to (Glu-urea-Lys)-PEG-DSG The addition of 2-PMPA caused a substantial inhibi-tion of (Glu-urea-Lys)-LNP uptake in LNCaP cells and little
or no effect on the uptake of nontargeted LNP (Figure 4a) Uptake of PSMA-targeted and untargeted LNPs was also measured in the PSMA-negative PCa cell line PC-3 (ref 38);
no enhancement in LNP uptake due to the presence of the (Glu-urea-Lys) ligand in the LNP would be expected In these cells, significantly greater LNP uptake was observed for non-targeted LNP compared to PSMA-non-targeted LNP (Figure 4b), possibly due to charge repulsion between the plasma mem-brane and the negatively charged (Glu-urea-Lys) targeting ligand
We next evaluated whether the presence of the (Glu-urea-Lys)-PEG-DSG would lead to enhanced target gene silencing
in LNCaP cells In LNCaP cells treated with PSMA-targeted
Figure 2 Systemic administration of lipid nanoparticle (LNP)
containing 5 mol% PEG-DSG results in greater accumulation
in LNCaP tumors compared to LNP containing 2.5 mol%
PEG-DSG (a) Mice were injected via the tail vein with fluorescently
labeled LNP containing 5 or 2.5 mol% PEG-DSG (red) and were
sacrificed 4 or 24 hours following the final i.v injection LNCaP
tumors were harvested, cryo-sectioned, and analyzed under a
confocal microscope Representative images are shown Nuclei
were stained with Hoescht (blue) (b) Quantitation of uptake of
fluorescent label into LNCaP tumor tissues was performed using
ImageJ (n = 5) (http://rsb.info.nih.gov/ij/); *P < 0.05; **P < 0.01.
2.5% PEG-DSG
5.0 % PEG-DSG
PBS
500
400
300
4 hours
24 hours
200
100
0
**
*
a
b
Trang 4LNP containing 0.5 or 1 mol% (Glu-urea-Lys)-PEG-DSG, AR
protein levels were significantly lower than in cells incubated
with nontargeted LNP (Figure 4c) In addition, a greater
inhi-bition of AR expression was observed with the formulation
containing 1 mol% (Glu-urea-Lys)-PEG-DSG than 0.5 mol%
(Glu-urea-Lys)-PEG-DSG
LNP-AR-siRNA systems containing the PSMA-targeting
Glu-urea-Lys ligand exhibit long circulation lifetimes
As described previously, LNP systems exhibiting long
circula-tion characteristics are essential to achieving enhanced
accu-mulation at tumor sites In this context, it was important to
establish that the presence of the (Glu-urea-Lys)-PEG-DSG
did not negatively impact the circulation lifetime of the
PEG-DSG LNP systems employed here This is of potential
concern since (Glu-urea-Lys)-PEG-DSG contains three
car-boxylic acid chemical groups at its hydrophilic terminal end
(Figure 3) The pKa values of these carboxylic acid groups
are predicted to be 3.11, 3.69, and 3.99 (Marvin, ChemAxon, http://www.chemaxon.com/products/marvin/), indicating that this PSMA-targeting ligand will possess a strong negative charge at physiological pH Consistent with these estimates, the zeta-potential of LNP containing 1 mol% of (Glu-urea-Lys)-PEG-DSG was determined to be −14.97 ± 9.34 mV, whereas nontargeted LNP exhibited a zeta-potential of
−4.91 ± 11 mV LNPs exhibiting negative charges can be rap-idly cleared from the bloodstream via opsonization by serum proteins and subsequent accumulation in the reticuloendo-thelial cells of the liver and spleen.39,40
The circulation lifetime of the PSMA-targeting LNP was determined following i.v administration of tritiated (3H) PSMA-targeted or nontargeted LNP to mice at 1 mg siRNA/
kg body weight (see Materials and Methods) Blood was col-lected via intracardiac sampling at 0.5, 2, 8, 24 hours postin-jection and the percentage of the injected LNP remaining in the circulation was determined (Figure 5) Importantly, the
Figure 3 Reagents and conditions for synthesis of (Glu-urea-Lys)-PEG-DSG: (i) (PPh3)2PdCl2, CuI, triethylamine/acetonitrile, 80 °C,
3 h, 95%; (ii) a N,N,N’,N’-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIEA)/dichloromethane (DCM), rt, overnight 93% and b H2, Pd-C/ methanol, rt, overnight, 92%; (iii) a N,N’-disuccinimidyl carbonate
(DSC), triethylamine, DCM, 0 °C to room temperature, overnight and b methyl 6-aminocaproate hydrochloride, pyridine/DCM, 0 °C to rt,
overnight, 88%; (iv) a LiOH, methanol/water/THF, 0 °C to rt, overnight, 96% and b N-hydroxysuccinimide,
N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDAC), 4- (dimethylamino) pyridine (DMAP)/DCM, rt, overnight; (v) Triethylamine (TEA)/DCM, rt, overnight,
66%; (vi) formic acid/DCM (2:1), rt, overnight, 91%; (vii) HBTU, DIEA / N,N-dimethylformamide (DMF), rt, overnight, 43%; (viii) neat formic
acid, 48%
HO
O
O
O
O O
O
O
O
O O O O O
O O n
O
O N
NH
NH
NH
NH O HOOC
HOOC
COOH NH NH
H
H
H
H
H
H
H
H H
O
O
O O
O
O O
O
O O
PSMA-targeting PEG-DSG
O O
H O
O O
H
NH
BocHN (PEG2000)
(PEG2000)
(PEG2000)
(PEG2000)
NH
MeO
(i)
(ii)
(iii)
(iv)
(v)
(vi)
10, R = Boc
11, R = H
(vii)
(viii)
13 12
5
RHN
n
n
n
1
2
4
3
7 6
8
9 +
Trang 5PSMA-targeted and nontargeted LNPs exhibited very
simi-lar circulation properties; both formulations had t1/2 values of
approximately 10–12 hours Since it was previously shown
that LNP-siRNA systems incorporating PEG-DSG
accumu-late in tumors (Figure 2),11 these data support the probability
that (Glu-urea-Lys)-LNP-siRNA will exhibit similar
accumula-tion at distal tumor sites
PSMA-targeted (Glu-urea-Lys)-LNPs enhance AR
knockdown in mice bearing LNCaP tumors
The potency of long-circulating, PSMA-targeted
(Glu-urea-Lys)-LNP systems was evaluated in athymic nude mice
bearing LNCaP tumors When serum PSA levels reached
50–75 ng/ml, mice were randomly assigned to three
experi-mental groups and treated i.v with phosphate buffered saline
(PBS) or PSMA-targeted or nontargeted LNP at a dose of
5 mg siRNA/kg body weight While serum PSA levels rose
to 40% above the pretreatment levels by Day 14 in the
con-trol group, PSA levels remained relatively unchanged
com-pared to baseline in mice treated with nontargeted LNP
(Figure 6a) This represents a significant improvement over
previous studies, since similar effects on PSA levels were
observed at half the dose used for the first generation
LNP-AR-siRNA.1 The enhanced potency can be attributed to the
combined effects of a more potent cationic lipid and a more
active siRNA payload, as well as greater LNP accumulation
at the distal tumor site due to enhanced circulation
char-acteristics Even more promising were the results from the
PSMA-targeted (Glu-urea-Lys)-LNP group; in these mice, a
45% reduction in the serum PSA levels was observed at day
14 compared to the control group (Figure 6a) In mice treated
with siRNA formulated in the PSMA-targeted
(Glu-urea-Lys)-LNP, a decrease in serum PSA levels relative to pretreatment
levels was observed To directly verify AR gene silencing,
lev-els of AR and PSA mRNAs were assessed in samples from
LNCaP tumors at day 14 via quantitative real-time
reverse-transcription polymerase chain reaction (qRT-PCR) In the
PSMA-targeting (Glu-urea-Lys)-LNP group, there was a
sig-nificant reduction in AR mRNA transcript levels compared to
mice treated with the nontargeted LNP or PBS (Figure 6b)
The siRNA formulated in the nontargeted LNP did not cause
a significant reduction in mRNA transcript levels compared
to the PBS control (Figure 6b) These results are consistent
with AR protein levels (Supplementary Figure S1) Similar
results were obtained when levels of PSA mRNA transcript
were evaluated There was a significant decrease in levels
of PSA mRNA in the PSMA-targeted LNP treatment group
compared to mice treated with nontargeted LNP-AR-siRNA
(Figure 6c)
Intravenous administration of PSMA-targeted (Glu-
urea-Lys)-LNP reduces cellular proliferation, but does
not enhance apoptosis
The ultimate goal in cancer treatment is to induce tumor
regression; this has never been observed in the LNCaP
xeno-graft model using gene-silencing strategies, even in
con-junction with complete androgen ablation via castration.1,30
Figure 4 Lipid nanoparticle (LNP) formulations containing (Glu-urea-Lys)-PEG-DSG enhances cellular uptake and inhibition
of AR expression in AR-positive LNCaP cells in vitro (a)
LNCaP cells were incubated for 24 hours with fluorescently labeled AR-targeting LNP at an siRNA concentration 5 μg/ml Cellular uptake was quantified using Cellomics ArrayScan and is expressed
as mean fluorescent intensity per cell Approximately 400 cells
were measured in four individual wells (n = 4) 2-PMPA was added
to compete with AR-dependent uptake Statistical significance was determined for (Glu-urea-Lys)-LNP in comparison to all the
other groups; **P < 0.01 (b) PC-3 (AR-negative) PCa cells were
incubated with 5 μg/ml of fluorescently labeled (Glu-urea-Lys)-LNP for 24 hours 2-PMPA was added as a competitor Cellular uptake was quantified using Cellomics ArrayScan and expressed as mean fluorescent intensity per cell Approximately 400 cells were
measured in four individual wells (n = 4) Statistical significance was
determined between non-targeted LNP versus PSMA-targeted
(Glu-urea-Lys)-LNP groups **P < 0.01 (c) LNCaP cells were incubated
with 1, 5, 10, or 15 μg/ml siRNA formulated as indicated for 48 hours Levels of AR protein were analyzed by immunoblotting, with β-actin
as the loading control
70
**
50 40 30
(Glu-urea-Lys)LNP (Glu-urea-Lys)-LNP + 2-PMPA Nontargeted LNP
Nontargeted LNP + 2-PMPA
20
Mean fluorescent intensity 10
(Glu-urea-Lys)-LNP
1.0% (Glu-urea-Lys)-PEG-DSG
0.5% (Glu-urea-Lys)-PEG-DSG
0% (Glu-urea-Lys)-PEG-DSG Nontargeted LNP
0
0
AR µg/ml siRNA
β-actin
10 20 30
40 50 60 70 80
Treatment
Treatment
a
b
c
Trang 6LNCaP tumors were isolated from mice treated with PBS as
a control or AR21-siRNA formulated in PSMA-targeted or
nontargeted LNPs on day 14 Tumors were sectioned and
analyzed by immunohistochemistry for cellular proliferation
as indicated by staining for Ki67 Ki67 is a marker that is
detected during all phases of the cell cycle, but is absent in
quiescent cells.41 Data indicated that treatment with
AR21-siRNA formulated in the PSMA-targeted
(Glu-urea-Lys)-LNP caused an approximate 50% decrease in Ki67-positive
cells relative to levels in tumors from PBS-treated mice
(Figure 7a,b) No effect on apoptosis was observed based
on TUNEL staining of LNCaP tumors from treated and
untreated animals (Figure 7c,d) The lack of apoptotic cells
may be due to the short duration of this study (14 days)
Discussion
In previous work, we showed that AR25-siRNA formulated in
LNP containing DLin-KC2-DMA and PEG-DMG (a PEG-lipid
that rapidly dissociates from the LNP following i.v injection)
inhibited expression of the AR gene in an animal model of
PCa The potential for translating this work into the clinic is
limited by the high doses necessary to achieve an
appre-ciable pharmacological effect Here, we describe the
opti-mization of properties of the LNP-AR-siRNA systems to
achieve improvements in the in vivo gene-silencing potency
An improved cationic lipid, DMAP-BLP, was used and the
siRNA sequence and chemistry were optimized In addition,
a PEG-lipid (PEG-DSG) that does not readily dissociate from
LNP systems and a PSMA-targeting lipid were incorporated,
resulting in a longer lifetime in circulation, improved
accumu-lation at distal tumor sites, and PSMA-mediated uptake into
prostate tumor cells
Inclusion of 5 mol% PEG-DSG clearly resulted in improved LNP accumulation at the tumor site compared to LNP with lower amounts of the PEG-lipid (Figure 2) This had a mini-mal impact on LNP-siRNA potency, as judged by PSA levels, compared to the previously reported work.1 However, although the more stable PEG coatings lead to improved tumor accu-mulation, the PEG coat can impede cellular uptake, reducing the activity of the LNP payload Ideally, the PEG-lipid would remain associated with the LNP until arrival at the tumor site, after which it would dissociate For actively targeted systems
Figure 5 PSMA-targeted (Glu-urea-Lys)-LNP and non-targeted
LNP exhibit similar pharmacokinetics PSMA-targeted
(Glu-urea-Lys)-LNP and nontargeted lipid nanoparticle (LNP) were
synthesized with trace amounts of [3H] CHE The LNP formulations
contained DMAP-BLP/DSPC/cholesterol/(Glu-urea-Lys)-PEG-DSG/
PEG-DSG molar ratios of 50/10/35/1/4 or 50/10/35/0/5 or
DMAP-BLP/DSPC/cholesterol/PEG-DSG at molar ratios of 50/10/37.5/2.5
Mice treated by tail vein injection with 1 mg/kg siRNA formulated
in PSMA-targeted (Glu-urea-Lys)-LNP or non-targeted LNP Plots
show percentage of the total injected dose remaining as a function of
time Each data point represents the mean ± SD (n = 4) Circulation
halftime (t1/2) for nontargeted 2.5% PEG-DSG LNP, nontargeted 5%
PEG-DSG LNP and (Glu-urea-Lys)-LNP was ~0.5, 8.7, and 11.6
hours, respectively Area under the curve (AUC) for nontargeted 2.5%
PEG-DSG LNP, nontargeted 5% PEG-DSG LNP and
(Glu-urea-Lys)-LNP was 309.18 ± 35.22%·hours, 1,066.57 ± 57.21%·hours,
and 1,095.90 ± 45.65%·hours, respectively
100
90
80
70
60
50
40
% Injected dose 30
20
10
0
Time (hours)
Nontargeted LNP (5% PEG-DSG total (Glu-urea-Lys)-LNP (5% PEG-DSG total) Nontargeted LNP (2.5% PEG-DSG total)
Figure 6 Systemic administration of siRNA formulated in PSMA-targeted (Glu-urea-Lys)-lipid nanoparticle (LNP) lowers serum PSA levels and enhances AR gene silencing Mice were
treated via tail vein injection with PBS as a control or with the 5 mg/
kg of siRNA formulated in PSMA-targeted (Glu-urea-Lys)-LNP or nontargeted LNP (a) Percentages of serum PSA levels are relative
to PSA levels one day prior to treatment Serum PSA levels were measured on days 7 and 14 (b) Quantitative real-time PCR was
used to assess AR mRNA levels from tumor tissue on day 14 (c)
Quantitative real-time PCR was used to assess PSA mRNA levels
from tumor tissue on day 14 Data points are the means ± SE (n = 6–7); **P < 0.01.
(Glu-urea-Lys)-LNP) 0
50
**
100 150 200
Nontargeted LNP
PBS
(Glu-urea-Lys)-LNP)
1.25
1.00
0.75
0.50
0.25
ct )
ct )
0.00
0.0 0.5 1.0 1.5
Nontargeted LNP
PBS
Day7
Day14
a
b
c
**
**
Trang 7such as those containing the PSMA-targeted
(Glu-urea-Lys)-PEG-lipid, it is possible that the presence of the PEG-lipid
may reduce the endosomolytic properties of the LNP
follow-ing ligand-dependent uptake
The results presented demonstrate significantly greater
reductions in AR (~40%) and PSA (~50%) mRNA when the
AR21-siRNA was formulated in the PSMA-targeted
(Glu-urea-Lys)-LNP system compared to the nontargeted LNP
system (Figure 6) The use of the small molecule targeting
ligand has a number of advantages compared with use of
larger entities such as antibodies; most notably, the small
molecule targeting ligands can be incorporated at the time
of LNP manufacture rather than after the LNP is formed and
well-defined, scalable systems are more readily achieved
It is perhaps surprising that the highly negatively charged
PSMA-targeting Glu-urea-Lys ligand adopted a configuration
when incorporated into the LNP in which it is available on the
external surface for ligand binding A concern was that the
targeting ligand could become associated with the cationic
lipid during formulation and is buried in the interior of the LNP
as a result It is possible that the PEG tether limits internal
localization of the (Glu-urea-Lys)-PEG-DSG due to polarity
and/or steric effects
The data presented here demonstrate that the potency of
LNP siRNA systems to silence the AR following i.v
adminis-tration can be improved from dose levels of 10 mg/kg siRNA
to 5 mg/kg when more potent cationic lipids, higher levels
of tumor accumulation and the PSMA targeting ligand is
employed However, the dose levels of 5 mg siRNA/kg body
weight required for AR silencing are still approximately an
order of magnitude too high for clinical applications to be
envisaged, and further work is required to improve potency
This is especially important for achieving tumor growth
regres-sion, as we observed no statistically significant difference
in the tumor sizes between PSMA-targeted LNP treatment mice and control groups, despite appreciable knockdown of the AR and PSA in the treatment group (Supplementary Figure S2) Ways forward include higher levels of external
PSMA-targeting lipid or extending the PSMA-targeting moi-eties beyond the PEG coat to improve targeting could be envisioned It has been noted that use of a PEG5000 tether
to extend the targeting ligand further from the LNP surface resulted in a 160-fold improvement in targeting capability
in vitro.42 Incorporation of an additional ligand that targets another cell surface factor, such as the prostate stem cell antigen,43 may synergistically enhance the LNP uptake Finally, nanoparticle systems smaller than 50 nm exhibit sig-nificantly improved delivery to tumor cells by virtue of their ability to achieve improved tumor penetration.44 Of relevance
to this approach, the microfluidic mixing technique does offer the possibility of manufacturing LNP with sizes as small as
20 nm in diameter,33 and efforts will be directed toward reduc-tions in the size of the LNP-siRNA system
The potential of targeting ligands is demonstrated by recent highly encouraging clinical data utilizing siRNAs directly
conjugated to a N-acetylgalactosamine (GalNAc) targeting
ligand which have demonstrated efficient systemic deliv-ery allowing robust and durable mRNA knockdown of vari-ous targets in hepatocytes.45 The GalNAc ligand targets the highly expressed ASGPR receptor in hepatocytes—which presents the important advantages of exhibiting high copy number and a quick turnover In this aspect, the selection of efficient receptors for targeting is a key parameter for the suc-cess of targeted siRNA systems In addition, recently devel-oped biodegradable lipids enabling rapidly eliminated LNPs that display improved tolerability and safety profiles can be incorporated into potent and safe LNP-siRNA systems.46 It is therefore expected that this continuous progress in the field
Figure 7 Systemic administration of PSMA-targeted (Glu-urea-Lys)-lipid nanoparticle decreases cellular proliferation but does not induce apoptosis in tumor cells Tissues from the mice described in Figure 6 were analyzed (a) Representative images of Ki67 (cell
proliferation marker) stained samples (b) Quantitation of Ki67 stained tumor sections plotted as means ± SD (n = 6); **P < 0.01 (c) Representative
images of TUNEL (cell apoptosis marker) stained samples, with red circles highlighting stained cells (d) Quantitation of TUNEL stained tumor
sections plotted as the means ± SD (n = 6) No significant difference was observed for TUNEL-stained tumor sections.
100
80
60
20
0
100
80
60
40
20
0
(Glu-urea-Lys)-LNP
Nontargeted LNP
(Glu-urea-Lys)-LNP
Nontargeted LNP
PBS
Non Non
Trang 8of siRNA medicines will lead to LNP-AR-siRNAs targeting
PCas that have direct clinical utility
Materials and methods
Materials 1,2-Distearoyl-sn-glycero-3-phosphocholine
(DSPC) was purchased from Avanti Lipids (Alabaster, AL),
cholesterol (Chol) was purchased from Sigma (St Louis, MO)
1,1′-Dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine
perchlo-rate (DiI) was bought from Invitrogen (Burlington, ON, Canada)
The ionizable cationic lipid DMAP-BLP and PEG-lipids
(R)-2,3-bis(octadecyloxy)propyl-1-(methoxy poly(ethylene glycol)
2000) carbamate (PEG-DMG) and (R)-2,3-bis(stearyloxy)
propyl-1-(methoxy poly(ethylene glycol)2000 carbamate
(PEG-DSG) were synthesized at Alnylam Pharmaceuticals
(Cambridge, MA) The PSMA inhibitor
2-(phosphonomethyl)-pentanedioic acid (2-PMPA) was purchased from Cedarlane
(Burlington, ON, Canada)
Cell culture, cell lines, and reagents LNCaP and PC-3
human PCa cell lines were used in all in vitro experiments.38,47
LNCaP and PC-3 cells were obtained from ATCC and were
not passaged beyond 6 months after receipt or
resuscita-tion LNCaP cells were maintained in RPMI 1640 (Life
Tech-nologies, Burlington, ON, Canada), supplemented with 10%
heat-inactivated fetal bovine serum PC-3 cells were
main-tained in DMEM (Life Technologies, Burlington, ON, Canada)
supplemented with 5% heat-inactivated fetal bovine serum
Both cell lines were incubated at 37 °C with 5% CO2
siRNA sequences The sequence of the human AR gene
(GenBank accession no NM_000044) was extracted from
the NCBI Entrez nucleotide database The AR21-siRNA
was composed of two complementary RNA strands: sense
strand (AR-S) 5′-cuGGGAAAGucAAGcccAudTsdT -3′ and
antisense strand (AR-AS):
5′-AUGGGCUUGACUUUCCcAG-dTsdT-3′ The two strands of the AR21-siRNA are modified
21-nt oligoribonucleotides that contain phosphorothioate
linkages (indicated as “s”) between the 3′-deoxythymidine
(dT) overhangs and that include multiple 2′-OMe
modifica-tions (indicated by lower-case letters)
Oligonucleotide synthesis Oligonucleotides were
synthe-sized using an ABI-394 DNA/RNA synthesizer Solvents/
reagents, solid-supports and phosphoramidites were all
purchased from Glen Research or ChemGenes and used
as received Oligonucleotides were synthesized using
modi-fied synthesis cycles provided with the instrument After solid
phase synthesis, the oligonucleotides were deprotected and
released from the support The crude oligonucleotides were
purified by anion-exchange high performance liquid
chroma-tography (HPLC) to >85% (260 nm) purity and then desalted
by size exclusion chromatography The isolated yields for the
final oligonucleotides were calculated based on the
respec-tive ratios of measured to theoretical 260 nm optical density
units (ODUs) and their identity was confirmed by LC/MS
Hybridization to generate double-stranded siRNA duplexes
was performed by mixing equimolar amounts of purified
com-plementary strands to a final concentration of 20 μmol/l in 1×
PBS buffer pH 7.4, and by heating the solution over a water bath at 95 °C for 5 minutes and cooling it to room tempera-ture over a period of approximately 12 hours
Chemical synthesis Detailed chemical synthesis
proce-dures and characterization data of all intermediates (accord-ing to Figure 3) is given as Supplementary Materials and Methods only.
Preparation of tris-(t-Butyl) protected
and Methods, 1.00 g, 0.37 mmol) and compound 11 (see Supplementary Materials and Methods, 0.35 g, 0.48 mmol) were dissolved in DMF (10 ml) under argon atmosphere HBTU (0.22 g, 0.58 mmol) and DIEA (0.250 ml, 1.46 mmol) were added to the mixture, and the mixture was stirred over-night The solvents were removed under reduced pressure, and the residue was purified by silica gel chromatography (5–20% MeOH in DCM) to yield compound 12 (0.53 g, 43%)
as a white solid 1H NMR (400 MHz, DMSO-d6): δ 7.78 (t, J
= 5.7 Hz, 2H), 7.14-7.05 (m, 3H), 6.26 (m, 2H), 4.11-3.83
(m, 5H), 3.75–3.63 (m, 3H), 3.60–3.30 (m, OCH2- and
O-CH-protons, PEG, O-alkyl and glycerol), 3.27–3.12 (m, 5H),
3.07–2.85 (m, 4H), 2.30–2.15 (m, 3H), 2.03 (t, J = 7.4, 2H), 1.59–1.33 (m, 23H), 1.30–1.18 (m, 60H), 0.84 (t, J = 6.7 Hz,
6H) MS calc Av MW ~ 3,343; MALDI Av MW found: 3,342
Preparation of (Glu-urea-Lys)-PEG-DSG (13) Formic acid
(20 ml) and anisole (0.5 ml) were added to compound 12 (0.50 g, 0.14 mmol), and the mixture was stirred at room temperature for 24 hours The solvents were removed under reduced pressure, and the residue was coevaporated with toluene (2 × 50 ml) The crude compound was purified by flash silica gel column chromatography using a gradient of 10–50% MeOH in DCM followed by MeOH to yield com-pound 13 as a white solid (230 mg, 48%) 1H NMR (400 MHz,
DMSO-d6): δ 7.96 (bs, 2H), 7.80–7.77 (m, 3H), 7.22–6.95 (m, 6H), 4.09–3.80 (m, 6H), 3.68–3.60 (m, 2H), 3.59–3.40
(m, OCH2- and O-CH-protons, PEG), 3.39–3.08 (m, 33H),
3.05–2.87 (m, 5H), 2.76–2.57 (m, 4H), 2.36–2.14 (m, 3H),
2.03 (t, J = 7.5 Hz, 5H), 1.78 (brs, 2H), 1.65–1.60 (m, 2H), 1.55–1.30 (m, 15H), 1.29–1.13 (m, 61H), 1.10–1.00 (m, J = 6.5 Hz, 19H), 0.84 (t, J = 6.7 Hz, 6H) MS calc Av MW ~
3,190; MALDI Av MW found: ~3,193
Encapsulation of siRNA into LNP using microfluidic mixing
LNP formulations were constructed using a microfluidic stag-gered herringbone micromixer (SHM) provided by Precision Nanosystems (Vancouver, BC) as described previously.33
The siRNA solutions were prepared in 25 mmol/l acetate buf-fer at pH 4.0 For comparison studies between AR21-siRNA and AR25-siRNA, lipid stocks were co-dissolved in ethanol at molar ratio of 40% DMAP-BLP, 17.5% DSPC, 40% Chol, and 2.5% PEG-DMG For PEG-lipid comparison studies contain-ing 2.5 mol% total PEG-lipid, lipid stocks were codissolved
in ethanol at appropriate molar ratios: 50 mol% DMAP-BLP, 10 mol% DSPC, 37.3 mol% Chol, 0/0.5/1 mol% (Glu- urea-Lys)-PEG-DSG, 2.5/2/1.5 mol% PEG-DSG, and 0.2 mol% DiI For cationic LNPs encapsulating AR21-siRNA
containing 5 mol% total PEG-lipid used for in vivo studies,
Trang 9lipid stocks were codissolved in ethanol at the following molar
ratios: 50 mol% DMAP-BLP, 10 mol% DSPC, 34.8 mol% Chol,
0/1 mol% (Glu-urea-Lys)-PEG-DSG, 5/4 mol% PEG-DSG,
and 0.2 mol% DiI The siRNA to lipid ratio for all formulations
was kept at 0.067 (wt/wt) The siRNA and lipid ethanol
solu-tions are mixed at a 1:3 ratio, respectively Total formulation
volume ranges from 4 to 40 ml depending on experiment size
Characterization of LNP The mean diameter of the vesicles
was determined using a NICOMP370 particle sizer (Nicomp
Particle Sizing, Santa Barbara, CA) Intensity-weighted size
and distribution data was used LNPs utilized for AR21-siRNA
and AR25-siRNA comparisons were 56.5 ± 17.6 nm and
55.9 ± 19.61 nm in size, respectively The size of LNP
contain-ing a total of 2.5 mol% PEG-lipid,
(Glu-urea-Lys)-LNP-AR21-siRNA (1 mol% (Glu-urea-Lys)-PEG) was 84.5 ± 32.5 nm,
LNP-AR21-siRNA (0.5 mol%
(Glu-urea-Lys)-PEG) was 77.5 ± 23.6 nm, and the non-targeted
LNP-AR21-siRNA (0 mol% (Glu-urea-Lys)-PEG) was 73.6 ± 31.6 nm For
LNPs containing a total of 5.0 mol% PEG-lipid:
(Glu-urea-Lys)-LNP-AR21-siRNA (1 mol% (Glu-urea-Lys)-PEG-DSG)
the size was 55.9 ± 22.5 nm, and the non-targeted
LNP-AR21-siRNA (no targeting PEG-lipid) exhibited a size of
45.3 ± 16.5 nm Zeta potentials of LNPs were measured using
the Malvern Nano ZS (Worcestershire, UK) Lipid
concen-trations were determined based on total cholesterol content
determined using the Cholesterol E enzymatic assay from
Wako Chemicals (Richmond, VA) Concentrations of siRNA
were measured using Quant-iT RiboGreen RNA Reagent
and Kit (Life Technologies) according to the manufacturer’s
protocol Encapsulation efficiency was determined by
analy-sis of siRNA concentrations after addition of 1% Triton-X-100
(Sigma) to intact LNPs
Western blotting LNCaP cells were plated in 12-well plates
(2.0 × 105 cells per well) Cells were washed with PBS and
lysed with RIPA buffer (1% NP-40, 0.25% deoxycholic acid)
supplemented with protease inhibitors (Roche Diagnostics,
Laval, Quebec, Canada) Aliquots of 10 μg of total protein, as
quantified by Bradford Assay, were analyzed by
immunoblot-ting Antibodies to AR were purchased from Santa Cruz
Bio-technology (AR-441) (Santa Cruz, CA) Antibodies to β-Actin
were purchased from Abcam (Cambridge, MA)
Antigen-antibody complexes were detected using Millipore Immobilon
Western Chemiluminescent HRP Substrate (Billerica, MA)
Confocal microscopy of tumor sections Excised LNCaP
tumors from groups of three mice each were maintained in
10% buffered formalin and then cryo-sectioned by Wax-IT
Histology Services (Vancouver, BC) Tissue sections were
fixed onto glass cover slips and examined under an
Olym-pus FV1000 (Center Valley, PA) laser-scanning microscope
For each mouse xenograft, 5 tissue sections and 20 fields of
view were examined LNP fluorescence was quantified using
Image J (v1.50b, https://imagej.nih.gov/)
Fluorescent microscopy LNCaP cells were seeded at 2.0 × 104
cells per well and PC-3 cells were seeded at 1.5 × 104 cells per
well in a 96-well format Cells were treated with 5 μg/ml (based
on AR21-siRNA weight) (Glu-urea-Lys)-LNP-AR21-siRNA (1
mol% (Glu-urea-Lys)-PEG-DSG) or LNP-AR21-siRNA for
24 hours 2-PMPA was added as a competitive reagent at 100-fold molar excess to (Glu-urea-Lys)-PEG-DSG Cells were fixed in 3% PFA with Hoechst’s stain and examined using a Cellomics ArrayScan VTI HCS Reader (Thermo Sci-entific, Pittsburgh, PA)
Pharmacokinetics of PSMA-targeted and nontargeted LNP-AR-siRNAs Female CD1 outbred mice (6 to 8 weeks old)
were obtained from Charles River Laboratories (Wilmington, MA) and acclimated for one week prior to use Mice were given 1 mg/kg of either PSMA-targeted or nontargeted LNP-AR21-siRNA, containing trace amounts of [3H]-cholesteryl hexadecylether (CHE), via the lateral tail vein injection At 0.5, 2, 8, and 24 hours postinjection, mice were euthanized Blood was collected via intracardiac sampling in Vacutainer tubes containing EDTA (BD Biosciences, Canada) and was chemically digested at room temperature using Solvable (Perkin-Elmer, Wellesley, MA) followed by decolorization with hydrogen peroxide (30% w/w) The amount of LNP in blood was determined by liquid scintillation counting in Pico-Fluor
40 (Perkin-Elmer) All procedures involving animals were approved by the Animal Care Committee at the University
of British Columbia and performed in accordance with the guidelines established by the Canadian Council on Animal Care
Treatment of mice with PSMA-targeted and non-targeted LNP-AR21-siRNAs Xenograft prostate tumors were
estab-lished as described.30 Briefly, LNCaP cells (5 × 106) in 0.1 ml Matrigel (Becton Dickinson Labware, Mississauga, Ontario, Canada) were inoculated subcutaneously in two flank regions of 6- to 8-week-old male athymic nude mice (Harlan Sprague Dawley, Indianapolis, IN) under halothane anes-thesia using a 27-gauge needle When the tumors became palpable, volumes were measured, and blood was collected from the tail vein to assess serum PSA by ELISA (ClinPro International, Union City, CA) Once PSA values reached 50–75 ng/ml, animals were randomized into three groups and were treated with (Glu-urea-Lys)-LNP-AR21-siRNA, nontargeted LNP-AR21-siRNA, or saline (PBS) via the tail vein Animals treated with PBS were given one injection on day 1 Animals given (Glu-urea-Lys)-LNP-AR21-siRNA and non-targeted LNP-AR21-siRNA were treated on days 1, 2,
3, 7, 9, and 11 with 5 mg siRNA/kg of mouse body weight Mice were sacrificed on day 14 Serum PSA levels and lev-els of mRNA encoding AR and PSA were determined, and immunohistochemical analyses of xenograft tumors were performed All animal procedures were performed according
to the guidelines of the Canadian Council of Animal Care and with appropriate institutional certification
qRT-PCR Total RNA from mouse tissue was isolated using
Trizol according to the manufacturer’s protocol (Life Technol-ogies, Burlington, ON, Canada) RNA extracts were reverse transcribed using random hexamers (Applied Biosystems, Foster City, CA) and MMLV reverse transcriptase (Invitrogen) Triplicates of the resulting cDNA were used as templates for quantitative real-time PCR on the Applied Biosystems 7900HT Fast Real-Time PCR System following the SYBR Green PCR
Trang 10Master Mix protocol as described previously.48 18S rRNA was
used as an endogenous control and relative quantitation was
determined using the comparative Ct (2-ΔΔCt) method Primer
sequences for AR were: sense 5′-GCAGGCAAGAGCACT
GAAGATA-3′ and antisense 5′-CCTTTGTGTAACCTCCCTT
GA-3′ Primers for PSA were: sense 5′-TGTGCTTCAAGG
TATCACGTCAT-3′ and antisense: 5′- TGTACAGGGAAGG
CCTTTCG-3′ Primers for 18S rRNA were: sense 5′-CGA
TGCTCTTAGCTGAGTGT-3′ and antisense 5′-GGTCCAAG
AATTTCACCTCT-3′
Immunohistochemistry of tumor tissues
Immunohistochem-istry (IHC) staining was conducted using a Ventana
auto-stainer model Discover XT (Ventana Medical System Oro
Valley, AZ) with an enzyme-labeled biotin streptavidin
sys-tem and solvent- resistant DAB Map kit The antibody used for
Ki67 was from Lab Vision Corporation, and was diluted 1:500
in 1X PBS The TUNEL, or apoptosis, study was done using
a TdT enzyme kit (Roche, Indianapolis, IN) IHC slides were
scanned on a Leica Digital Imaging System Images were
viewed using Digital Image Hub, (SlidePath, Dublin, Ireland)
The proliferation factor is defined as the average number of
Ki67 positive cells per core, or per section The apoptotic
fac-tor is the average number of apoptotic positive cells per core,
or per section
Statistical analyses All statistical analyses were performed
using GraphPad Initially, a one-way analysis of variance was
used to statistically evaluate the differences between
treat-ment groups In the case of statistically significant results,
the differences between treatment groups were assessed
through the use of the Tukey-Kramer multiple comparisons
test, unless otherwise stated Probability (P) values less than
0.05 were considered significant
Supplementary material
Figure S1 Systemic administration of siRNA formulated in
PSMA-targeted (Glu-urea-Lys)-LNP enhances AR protein
knockdown in vivo
Figure S2 Effect of systemically administered LNP
formu-lations on tumor growth
Figure S3 LNP encapsulating scramble (SC)-siRNA does
not result in AR knockdown in vitro
Figure S4 siRNA against GAPDH does not result in AR
knockdown in vitro
Materials and Methods
Acknowledgments The authors would like to
acknowl-edge the Canadian Institutes of Health Research grants
(MOP-86587, FRN-111627, FRN-124295), Prostate Cancer
Canada grant (D2013-5) and Alnylam Pharmaceuticals for
supporting this work
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